Simple model organisms such as the free-living soil nematode C. elegans are experimentally tractable systems that can be used to provide insights into human development and disease. For example, genes associated with the development of cancer in humans have also been found in C. elegans. One of these human proto-oncogenes, termed TANT-1 (Ellisen et al., 1991), encodes a protein of the LIN-12/Notch family. This family was first identified by contemporaneous studies of the lin-12 gene in C. elegans and the Notch gene in Drosophila. It has been established that activating TAN-1 or a similar murine protein, Notch4, contributes to cancer formation. In C. elegans, activating LIN-12 affects cell fate decisions (Greenwald, e al., 1993; Greenwald and Seydoux, 1990; Struhl et al., 1993). Features common to all LIN-12/Notch proteins and their functions can be studied in C. elegans, and the results can be directly applied to mammals, with particular relevance to the study of cancer.
C. elegans can similarly serve as a model for processes involved in the development of Alzheimer's disease in humans. Two genes identified in linkage studies in humans encode related multipass transmembrane proteins, presenilins 1 and 2 (PS1 and PS2). The normal role of presenilins, and the mechanism by which mutant presenilins cause Alzheimer's disease, are not known. Genetic studies of the C. elegans presenilin SEL-12 (Levitan and Greenwald, 1995; Levitan et al., 1996) offer a powerful approach to understanding the normal role of presenilins. The basic biology of presenilins and Notch proteins is linked in both C. elegans and people: based on genetic interactions with lin-12, sel-12 has been shown to facilitate lin-12 signaling in C. elegans, and null mutations in the mouse PS1 and Notch1 genes have similar phenotypes (Wong et al., 1997; Shen et al., 1997).
The following is a detailed introduction to the first set of experiments, in which the sel-10 gene was identified as a regulator of lin-12 activity. In the first set of experiments, genetic interactions between sel-10 and lin-12 were discovered, and molecular and biochemical experiments were performed to elucidate the nature of the interaction. In a second set of experiments, interactions between sel-10 and sel-12 were discovered, consistent with the hypothesis that sel-10 possibly regulates sel-12 activity.
Many cell-cell interactions that specify cell fate are mediated by receptors of the LIN-12/Notch family and ligands of the Delta/Serrate/LAG-2 (DSL) family (reviewed Artavanis-Tsakonas et al., 1995). C. elegans affords an opportunity to study a simple case of lateral specification involving an interaction between two cells of the hermaphrodite gonad. These cells, named Z1.ppp and Z4.aaa, are initially equivalent in their developmental potential: each has an equal chance of becoming the anchor cell (AC), a terminally differentiated cell type that is necessary for vulval development, or a ventral uterine precursor cell (VU), which contributes descendants to the ventral uterus. However, in any given hermaphrodite, only one of these cells will become the AC, while the other becomes a VU (Kimble and Hirsh, 1979).
Laser ablation studies have shown that this process of lateral specification, the AC/VU decision, depends on interactions between Z1.ppp and Z4.aaa (Kimble, 1981; Seydoux and Greenwald, 1989). Furthermore, genetic studies have indicated that lin-12-mediated signalling controls the AC/VU decision: if lin-12 activity is inappropriately elevated, Z1.ppp and Z4.aaa become VUs, while if lin-12 activity is reduced, Z1.ppp and Z4.aaa become ACs (Greenwald et al., 1983). Genetic mosaic analysis (Seydoux and Greenwald, 1989) and reporter gene studies (Wilkinson et al., 1994) have indicated that both Z1.ppp and Z4.aaa initially express lin-12 and lag-2, but that a stochastic small variation in ligand and/or receptor activity is subsequently amplified by a feedback mechanism that influences lin-12 and lag-2 transcription. Thus, Z1.ppp and Z4.aaa assess their relative levels of lin-12 activity as part of the decision-making process, before either cell commits to the AC or VU fates, and the feedback mechanism ensures that only one of the two cells will become an AC and the other will become a VU.
It is striking that the receptors (lin-12/Notch proteins), ligands (DSL proteins), and at least one downstream signalling component (CBF1/Su(H)/LAG-1; see Christensen et al., 1996 and references therein) that mediate lateral specification are highly conserved in animals as distantly related as C. elegans, Drosophila, and vertebrates. Furthermore, a feedback mechanism like that first described for the AC/VU decision (Seydoux and Greenwald, 1989) also exists for a Notch-mediated lateral interaction in Drosophila (Heitzler and Simpson, 1991) and seems likely to operate in Notch-mediated lateral interactions in vertebrates (Austin et al., 1995; Chitnis et al., 1995; Washburn et al., 1997). The identification of genes that influence lin-12 activity during the AC/VU decision may reveal other conserved factors that participate in signal transduction or regulate the activity of lin-12/Notch proteins.
Genetic screens based on suppression or enhancement of lin-12 mutations have identified a number of genes that influence lin-12 activity. Here, sel-10 is described. It was first identified in a screen for suppressors of phenotypes associated with partial loss of lin-12 activity (Sundaram and Greenwald, 1993). sel-10 acts as a negative regulator of lin-12 signalling, and SEL-10 is a member of the CDC4 family of F box/WD40 repeat containing proteins. CDC4, the most extensively studied member of this family, is a Saccharomyces cerevisiae protein that is involved in the ubiquitin-mediated degradation of cell cycle regulators (reviewed in King et al., 1996).
The similarity of SEL-10 to CDC4 prompted investigation of the possibility that SEL-10 is involved in the ubiquitin-dependent turnover of LIN-12/Notch proteins. The experiments involved examining the biochemical effects of coexpressing C. elegans SEL-10 with a vertebrate LIN-12/Notch protein, Notch4. This vertebrate Notch gene was originally termed int-3, because it was identified by mouse mammary tumor virus insertions into a cellular gene (Gallahan and Callahan, 1989). In int-3 mutants, the viral long terminal repeat promotes expression of a truncated transcript that encodes a protein similar to the intracellular domains of LIN-12/Notch proteins (Robbins et al., 1992). The complete sequence of the gene defined by int3 revealed that the extracellular domain of the predicted protein also has the hallmarks of LIN-12/Notch proteins, and hence the gene is now known as Notch4 (Uyttendaele et al., 1996). During normal development, Notch4 expression is restricted primarily to endothelial cells (Uyttendaele et al., 1996). In int-3 mutants, the inappropriate expression of a truncated transcript encoding the intracellular domain of Notch4 in mammary epithelia may alter stem cell fate decisions, thereby contributing to the development of cancer. Furthermore, at least one human cancer, T cell acute lymphoblastic leukemia, has been associated with expression of a comparable truncated Notch protein (Ellisen et al., 1991), suggesting that inappropriate Notch activity could contribute to the development of a variety of tumors.
C. elegans SEL-10 physically interacts with murine Notch4 and causes a reduction in the steady-state levels of the murine Notch4 intracellular domain. Results suggest that the negative regulation of LIN-12/Notch by SEL-10 is an evolutionarily conserved feature, given the striking parallels between the effect of sel-10 activity on lin-12 in C. elegans and the effect of SEL-10 expression on Notch4 stability in mammalian tissue culture. Furthermore, the role of vertebrate Notch genes in oncogenesis suggests that vertebrate sel-10 counterparts may behave as tumor suppressors.